MiR-1976/NCAPH/P65 axis inhibits the malignant phenotypes of lung adenocarcinoma

Lung adenocarcinoma (LUAD) is a malignancy with an abysmal survival rate. High metastasis is the leading cause of the low survival rate of LUAD. NCAPH, an oncogene, is involved in the carcinogenesis of LUAD. However, the regulation of NCAPH in LUAD remains controversial. In this work, we identified an up-regulation of NCAPH in LUAD tissues. Patients who expressed more NCAPH had shorter overall survival (OS). Furthermore, NCAPH overexpression promoted LUAD cell migration while inhibiting apoptosis. MiR-1976 and miR-133b were predicted to target NCAPH expression by searching TargetScan and linkedomics databases. Following that, we confirmed that miR-1976 suppressed NCAPH by directly targeting a 7-bp region of NCAPH 3′ untranslated regions (UTR). In addition, increased expression of miR-1976 decreased the proliferation & migration and promoted apoptosis of LUAD cells, and the re-introduction of NCAPH reversed these influences. Furthermore, the xenograft and metastasis mouse models also confirmed that miR-1976 inhibited tumor growth and metastasis in vivo by targeting NCAPH. Finally, we found that MiR-1976 targeting NCAPH blocked the activation of NF-κB. In conclusion, miR-1976 inhibits NCAPH activity in LUAD and acts as a tumor suppressor. The miR-1976/NCAPH/NF-κB axis may, in the future, represent crucial diagnostic and prognostic biomarkers and promising therapeutic options.


Real-time PCR analysis
Total RNA was extracted from cells using a complete kit for RNA extraction (Tiangen Biotech, Beijing, China).The PrimeScript RT kit (TaKaRa, Japan) reverse-transcribes total RNA into first-strand complementary DNA.Real-time PCR was carried out on a Bio-Rad iQ5 machine using the SYBR Green PCR Kit (TaKaRa, Japan).The primers for NCAPH and β-actin are as follows : NCAPH: Forward,5'-ACA GTG CCT CCT CTC CTT CA-3' , Reverse,5'-CCG CTC CTT CTC ATC GTC AT-3' .Each sample was tested in triplicate.The relative expression levels of each gene were calculated using the 2-ΔΔCt method.

Cell migration assay
The suspension cells (3 × 10 4 ) in serum-free DMEM were infused into Transwell chambers.The Transwell chamber was cleaned with PBS after 48 h, fixed for 20 min with methanol, stained with 0.5% crystal violet, rinsed for 20 min with PBS to clean the background, dried, and photographed after sealing.

Cell apoptosis and cell cycle assay
The trypsinized A549/NCI-H1975 cells were adjusted to 3 × 10 5 cells/mL with serum-containing DMEM/RPMI-1640 medium.The Annexin V-APC and the 7-AAD Apoptosis Detection Kit (MultiSciences, China) were used in the cell apoptosis assay.The A549 or NCI-H1975 cells were transfected with miR-1976 mimic, miR-1976 inhibitor, Lv-NCAPH lentivirus, or sh-NCAPH lentivirus for 24 h.Then, the cells were treated with serum-free DMEM/RPMI-1640 medium for 24 h to induce apoptosis.Next, the cells were stained with Annexin V-APC and 7-AAD for 30 min in the dark.Cell cycle detection was performed using cell cycle staining buffer (MultiSciences, China).Cell cycle detection was conducted when the treated cells were washed with PBS and then stained with cell cycle staining buffer for 30 min.The samples were finally tested using a flow cytometer(Becton Dickinson), and the apoptosis populations were calculated by FlowJo X software.Similarly, the cell cycle was analyzed using FlowJo X software.

Laser confocal
A cell slide was first laid on a 12-well cell culture plate, and 1 × 10 5 cells/holes were inoculated.After 24 h of culture, the cells on the slide were stained with NF-κB activation-nuclear transport detection kit (Beytime, China), and then the slide was sealed on pathological tissue slides.The image was captured using a laser scanning microscope (Zeiss) to detect the fluorescence of DAPI and Cy3 under a 40-x oil lens.

CCK8 assay
In 96-well plates, LUAD cell groups were seeded.Each 96-well plate well was seeded with 500 cells per 100 μL.Replaced with fresh medium, ten microliters of CCK8 reagent were added to each well.Absorbance was measured at 450 nm in each 96-well plate after 24, 48, 72, and 96 h.

Xenograft assay and metastasis assay in vivo
The Experimental Animal Ethics Committee of Guilin Medical university has reviewed and approved all animal experiments (No.GLMC-IACUC-2023005).This study is reported in accordance with ARRIVE guidelines.Animal Technology Vital River Lab (Beijing, China) supplied male BALB/c nude mice.In vivo xenograft assay, 1 × 10 7 A549 cells stably transfected with control lentivirus, miR-1976 lentivirus, Lv-NCAPH lentivirus, miR-1976 lentivirus + Lv-NCAPH lentivirus were subcutaneously injected into mice, respectively.Naked mice were euthanized under anesthesia after four weeks of injection.The tumor was removed and weighed.In the metastatic assay, naked mice were injected with 1 × 10 7 A549 cells.After four weeks of injection, these mice were euthanized under anesthesia, and the lungs were removed and dyed using Bouin's solution.For euthanasia, each animal was euthanized with an overdose of sodium pentobarbital.We confirmed that all methods are carried out in accordance with the relevant guidelines and regulations.

Statistical analysis
The statistical analyses used GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) and SPSS v.25.0 (SPSS Inc., Chicago, IL, USA).The t-test was utilized to compare the two groups.One-way ANOVA was used to compare more than two groups.Correlations between clinical pathological variables (age, gender, TNM stages, T stage, N stage, M stage, and anatomic location) and NCAPH expression were assessed using Fisher's exact and Mann-Whitney U tests.P values were always two-sided, and a statistically significant P = 0.05 was used.The Kaplan-Meier with Log-rank (Mantel-Cox) test was utilized to calculate a survival analysis.

NCAPH is up-regulated in LUAD and associated with poor prognosis in patients with LUAD
To identify the potential biomarkers for LUAD, we first searched RNA-seq datasets from the GEO and the TCGA database.The TCGA data indicated that the level of NCAPH mRNA in LUAD tissues was significantly higher than that in normal tissues (Fig. 1A, P < 0.001).Similarly, GEO dataset GSE10072 (Fig. 1B, P < 0.001) and GEO dataset GSE43458 (Fig. 1C, P < 0.001) also showed NCAPH mRNA was up-regulated in LUAD samples as compared to normal controls.We further detected NCAPH mRNA levels in 14 LUAD and adjacent standard lung specimen pairs.We confirmed that the NCAPH mRNA level was significantly higher in LUAD tissues compared to those of the noncancerous counterparts (Fig. 1D, P < 0.001).In the 14 pairs of LUAD tissues, NCAPH expression was detected by IHC, showing that the level of NCAPH in LUAD tissues was higher than in normal tissues.(Fig. 1E, P < 0.001).Furthermore, we investigated the predictive significance of NCAPH in LUAD using the TCGA dataset.Using X-tile software, LUAD patients were split into two groups: those with high expression (n = 118) and those with low expression (n = 349).Patients with high NCAPH expression had a poorer overall survival rate than those with low NCAPH expression, according to the Kaplan-Meier survival curve (HR = 0.4933, 95% CI: 0.3544-0.6866,P = 0.0005) (Fig. 1G).

NCAPH promotes proliferation &migration and inhibits apoptosis of LUAD cells
Based on the findings, we investigated the effect of NCAPH on cell proliferation, migration, and apoptosis in LUAD cell lines (A549, NCI-H1975).Enhancement or silencing of NCAPH expression was performed by transfecting NCAPH knockdown/overexpression lentivirus into A549 and NCI-H1975, respectively (Fig. 2A-D).We constructed three recombinant lentiviruses directed to NCAPH and identified that sh-NCAPH#1 transfection obtained the most significant inhibitory rate for the NCAPH expression in LUAD cells (Fig. 2C,D).Therefore, sh-NCAPH#1 was used to perform the follow-up examination.The CCK8 assay revealed that Lv-NCAPH increased LUAD cells' proliferation activity compared to Lv-control cells (Fig. 2E,G, P <0.001, P < 0.05).The sh-NCAPH reduced the proliferation activity of LUAD cells (A549, NCI-H1975) compared to the sh-control (Fig. 2F,H, P <0.001, P < 0.05).NCAPH overexpression resulted in a significant enhancement in LUAD cell viability.The transwell assays showed that NCAPH overexpression promoted LUAD cell migration (Fig. 2I,J, P <0.001), and NCAPH silencing inhibited the migration in A549 and NCI-H1975 cells (Fig. 2I,J, P <0.001).Meanwhile, increased NCAPH inhibited LUAD cell apoptosis, while decreased NCAPH had the opposite effect (Fig. 2K,L, P < 0.05).On the other hand, the EdU staining found that Lv-NCAPH increased DNA replication activity in LUAD cells compared to Lv-control cells.The sh-NCAPH decreased the DNA replication activity of LUAD cells compared with sh-control (Fig. 3A, P < 0.01).Flow cytometry showed that Lv-NCAPH increased the distribution of S-phase LUAD cells compared with Lv-control cells.The sh-NCAPH decreased the distribution of S-phase LUAD cells compared with sh-control (Fig. 3B, P < 0.05).

MiR-1976 targets NCAPH expression
In recent studies, oncogenes such as NCAPH can be targeted by some microRNAs to participate in tumor occurrence.We looked through two databases, TargetScan, and linkedomics, for potential microRNAs targeted NCAPH.The Venn diagram displayed that both databases predicted that miR-1976 and miR-133b could target NCAPH expression (Fig. 4A).To evaluate the potential clinical relationship between NCAPH mRNA expression and miR-1976 and miR-133b, we first validated miR-1976, miR-133b and NCAPH mRNA levels in 14 paired patient samples by real-time PCR.The findings demonstrated that LUAD tissues had lower miR-1976 and miR-133b levels than the nearby normal tissues (Fig. 4B, P < 0.05, Fig. 4C, P < 0.001).Additionally, the correlations between the two microRNAs and NCAPH were investigated using Pearson's correlation test.NCAPH mRNA  We also verified the above results in the TCGA database.We used 519 LUAD tissues and 46 normal tissues to detect the expression of miR-1976.As mentioned above, there was less miR-1976 in LUAD tissues than in healthy tissues (Fig. 4F, P < 0.001).The TCGA data also confirmed the negative correlation of miR-1976 level with NCAPH mRNA level (Fig. 4G, R = − 0.228, P < 0.01).Additionally, using the TCGA dataset, we found that

The miR-1976 inhibits tumor growth in vivo by targeting NCAPH
To verify the above data in vivo, we further established the Xenograft model by subcutaneously implanting A549 cells into BALB/c immune-deficient mice for four weeks.We discovered that the tumor volume of the miR-1976 lentivirus group decreased compared to the control lentivirus group.In contrast, the tumor volume of the Lv-NCAPH lentivirus group considerably increased (Fig. 7A,B, P < 0.01).However, the tumor volume in the miR-1976 + Lv-NCAPH lentivirus group regained the control level.(Fig. 7A,B, P < 0.05).The tumor weight had similar findings (Fig. 7C, P < 0.05).These findings suggested that overexpressing miR-1976 could reduce the growth-promoting effects of NCAPH in vivo.
Then the total proteins of the tumor tissues in nude mice were extracted, and Western blot assays were used to find NCAPH protein expressions in all of the groups mentioned above.Compared to the control group, a significant down-regulated NCAPH protein level was observed in the miR-1976 high-expression group.NCAPH level was significantly up-regulated in the Lv-NCAPH lentivirus group compared to the control group (Fig. 7D,E, P < 0.001).The re-introduction of Lv-NCAPH lentivirus partially reversed the negative-regulation effect of miR-1976 on NCAPH expression (Fig. 7D,E, P < 0.001).Changes in NCAPH and cell proliferative and apoptotic indicators were also discovered using immunochemistry labeling in tumor samples.(Fig. 7F).As shown in Fig. 6F-G, NCAPH expression was lower in the miR-1976 over-expression group compared to the control lentivirus group.In contrast, NCAPH-positive cells were increased in the Lv-NCAPH lentivirus group (Fig. 7G, P < 0.001).Simultaneously, the PCNA expression showed the same trends as NCAPH protein expression (Fig. 7H, P < 0.001), while cleaved-caspase-3 displayed the opposite trends to NCAPH protein expression (Fig. 7I, P < 0.001).Notably, Lv-NCAPH + miR-1976 lentivirus co-transfection restored the expression of NCAPH, PCNA, and cleaved-caspase-3 to the control levels (Fig. 7G,H,I, P < 0.001).

The mir-1976 inhibits tumor metastasis by targeting NCAPH in vivo
To further comprehend how miR-1976 affected LUAD metastasis, we injected A549 cells into the tail vein to generate a lung cancer metastasis model.The findings revealed that the number of tumor nodules was significantly lower in the miR-1976 lentivirus group than in the control group.The number of nodules considerably increased in the NCAPH lentivirus group compared to the control (Fig. 7J,K, P < 0.001).The effect of the mir-1976-induced decrease in the number of tumor nodules was considerably reversed by the overexpression of NCAPH (Fig. 7J,K, P < 0.001).The HE staining experiment produced identical results (Fig. 7L).The studies above revealed that miR-1976 reduced LUAD metastases by in vivo targeting NCAPH expression.

MiR-1976 targeting NCAPH blocks the activation of NF-kB
Studies have revealed that the NF-kB pathway involves cell proliferation, migration, and other biological processes.To further explore the downstream pathway induced by the miR-1976/NCAPH axis, we detected the activation of NF-kB.The expression of phosphorylated p65 was shown to be up-regulated by NCAPH overexpression and down-regulated by NCAPH inhibition (Fig. 8A,B, P < 0.001).Despite NCAPH overexpression and knockdown, the overall level of p65 protein remained the same (Fig. 8A,B, P  www.nature.com/scientificreports/results were confirmed in the NCI-1975 cell line (Fig. 8C,D, P < 0.001).In addition, laser confocal analysis of LUAD cells showed that P65 significantly entered the nucleus from cytoplasm after overexpression of NCAPH (Fig. 9A).Notably, compared to the control, the overexpression of miR-1976 reduced the phosphorylation of p65.Meanwhile, the overexpression of miR-1976 could counteract an increase in phosphor-p65 level caused by the overexpression of NCAPH.(Fig. 8E-H, P < 0.05, P < 0.01, P < 0.001).Similarly, laser confocal showed that overexpression of miR-1976 significantly reduced NCAPH-induced P65 entry into the nucleus from cytoplasm (Fig. 9B). Vol.:(0123456789)

Discussion
Lung cancer is one of the most aggressive malignancies threatening human life and health 24 .As the population has become older, environmental pollution has gotten worse, and more people are smoking, the prevalence of lung cancer has been rising annually 25 .Surgery is the primary treatment option for early-stage lung cancer 26 .The onset of lung adenocarcinoma is insidious, and the early symptoms are atypical, so many patients miss the best treatment opportunity when they are diagnosed 27 .Recently, significant advances have been made in the molecular genetics and immunotherapy of lung adenocarcinoma, which brings new promise to patients suffering from lung adenocarcinoma 28 .Till now, NCAPH, as an oncogene, has been implicated in multiple dangerous cancer, such as breast cancer 7 , endometrial cancer 8 , and NSCLC 9 .In this study, we found that the expression of NCAPH was up-regulated in lung adenocarcinoma compared to standard control based on the TCGA and GEO database (Fig. 1A-C).Meanwhile, 14 LUAD specimens had significantly greater NCAPH mRNA levels and protein expression than comparable neighboring tissues (Fig. 1D,E,F).Moreover, lung cancer patients' survival was strongly correlated with the expression of NCAPH (Fig. 1G).According to recent investigations, NCAPH appears to be involved in various tumor cell biological activities.In pancreatic cancer (PC) cell lines, the down-regulated NCAPH inhibited PC cell proliferation and colony formation.NCAPH also plays a vital role in cell cycle progression and DNA damage.NCAPH downregulation induced cell apoptosis via a caspase-dependent Pathway 29 .NSCLC cells' invasion, migration, and colony formation were decreased by NCAPH knockdown, which also prevented their proliferative growth and caused a G2/M cell cycle stop.NCAPH was associated with the development and progression of lung adenocarcinoma and represented a potential therapeutic target in lung adenocarcinoma 29,30 .Our current research showed that overexpressing NCAPH greatly encouraged proliferation and migration and prevented apoptosis in LUAD cells (Fig. 2E-L).These findings revealed that NCAPH plays an oncogenic role in LUAD.In addition, we also found that NCAPH can promote DNA replication activity and cell enrichment in the S phase of LUAD cells (Fig. 3).In the study of Li et al. it was also found that inhibiting the expression of NCAPH could reduce the proliferation of lung adenocarcinoma cells, which is consistent with our findings 31 .However, how NCAPH is involved in the progression of lung adenocarcinoma remains unclear.
Our following question is why and how NCAPH is increased in LUAD tissues.Several studies have found that the miRNA-mRNA regulatory network is linked to carcinogenesis and survival prognosis in various cancers.Because the levels of miRNAs differ significantly between normal and malignant tissues, many of them can be employed as biomarkers for prognosis and prediction 32 .We hypothesized that microRNAs could alter NCAPH post-transcriptionally.Several miRNAs are involved in complicated regulatory networks.Many miRNAs can control a single gene, and a single miRNA can target multiple candidates 33 .Previous research found that miRNA-133b inhibited osteosarcoma cell motility, migration, invasion, and EMT by targeting FGFR1 34 .In NSCLC, miR-133b targeting NCAPH boosted β-catenin degradation while decreasing cancer stem cell maintenance 35 .Gang Chen et al. discovered that miR-1976 functioned as a tumor suppressor in the evolution of NSCLC and directly targeted PLCE1 23 .The TargetScan and linkedomics datasets were combined in our investigation to identify the potential microRNAs miR-1976 and miR-133b that target NCAPH (Fig. 4A).Real-time PCR revealed that miR-1976 and miR-133b levels in LUAD tissues were lower than in nearby normal tissues (Fig. 4B,C).Furthermore, Pearson's correlation test revealed a strong negative association between NCAPH mRNA levels and miR-1976 expression (Fig. 4D,R= − 0.8154, P < 0.001).However, miR-133b level was positively correlated with NCAPH mRNA level (Fig. 4E, R = 0.8758, P < 0.001).The TCGA data also confirmed the negative correlation of miR-1976 level with NCAPH mRNA level (Fig. 4G, R = − 0.228, P < 0.001).In addition, we used the TCGA dataset to explore the prognostic value of miR-1976.The result showed that the prognosis of patients with high expression of miR-1976 (n = 229) was significantly better than that of patients with low expression of miR-1976 (n = 231) (Fig. 4H).
Then, we further found that miR-1976 inhibited the expression of NCAPH.However, the NCAPH mRNA level could not be regulated by miR-133b (Fig. 5A,B).We designed luciferase reporter assays to explore further which part of NCAPH is targeted by miR-1976 to impair the NCAPH expression.We first predicted three binding sites of NCAPH 3 ′UTR region, which might be targeted by miR-1976 using the Targetscan database.We named them binding site1, binding site2, and binding site 3, respectively (Fig. 5E).We found only binding site 2 (1627 bp-1633 bp sites of NCAPH 3'-UTR) was targeted by miR-1976(Fig.5F-H), that is miR-1976 downregulated NCAPH expression by binding to the 1627 bp-1633 bp of NCAPH 3'UTR.Next, by constructing a mutant of binding site 2 and co-transfecting lung adenocarcinoma cells with miR-1976, it was found that miR-1976 could targeting NCAPH in vivo (Fig. 6).Following that, we discovered that the mir-1976/NCAPH axis plays a critical role in tumor growth and metastasis (Fig. 7) in mouse models.Xiong et al. have shown that miR-133b targeted NCAPH and performed a critical role in the progression of NSCLC 35 .However, we found that miR-1976, but not miR-133b, targeting NCAPH, was involved in lung adenocarcinoma's growth, metastasis, and apoptosis.
It is understood that NF-κB is a crucial participant in many stages of cancer development and progression 36,37 .We found that the overexpression of miR-1976 reduced the phosphorylation of p65, one of the NF-κB activation  , 9).According to the findings, miR-1976 selectively targeted NCAPH and then inhibited NF-κB activation.We attempt to explore how NCAPH promoted the phosphorylation of p65.We assumed that NCAPH might bind to IκB, an NF-κB inhibitor, decreasing its inhibitory activity and ultimately increasing p65 phosphorylation.Unfortunately, the Co-IP results revealed that NCAPH could not bind to IκB (supplementary Fig. 1A, B).In addition, NCAPH was not found to interact with P65 (supplementary Fig. 1C, D).The CoIP-MS (mass spectrometry) analysis will be undertaken to discover how NCAPH stimulates p65 phosphorylation.

Figure 1 .
Figure 1.NCAPH is up-regulated in LUAD and is associated with poor prognosis in patients with LUAD.Analysis of NCAPH mRNA expression in the TCGA dataset (A), GEO dataset GSE10072 (B), and dataset GSE43458 (C).NCAPH mRNA expression was detected in 14 paired human LUAD samples (D).IHC was used to detect NCAPH expression in LUAD tissues and adjacent normal tissues (P < 0.001) (E, F).In the TCGA database, Kaplan-Meier survival analysis was used to determine the prognostic significance of NCAPH expression (G).* P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3 .
Figure 3. NCAPH promotes the DNA replication activity and distribution during the S phase in LUAD cells.EdU staining was used to detect DNA replication activity in LUAD cells (A).Flow cytometry was used to detect the distribution of LUAD cells in the cell cycle (B).*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4 .
Figure 4. MiR-1976 targets NCAPH expression.MiR-1976 and miR-133b targeting NCAPH were identified using the TargetScan and linkedomics databases (A).The levels of miR-1976 and miR-133b were determined using real-time PCR in 14 pairs of clinical tissues (B, C).In 14 human LUAD tissues, the connections between miR-1976 levels and NCAPH mRNA levels (D) and between miR-133b levels and NCAPH mRNA levels(E) were examined using Pearson's correlation.The TCGA data for miR-1976 levels in LUAD and normal tissues were examined (F).Pearson's correlation was used to examine the relationship between miR-1976 levels and NCAPH mRNA levels in the TCGA database (G).The Kaplan-Meier was used to assess the prognostic value of miR-1976 (H).* P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 8 .
Figure 8. MiR-1976 targeting NCAPH blocks the activation of NF-kB.The A549 cells and NCI-H1975 cells were infected with the corresponding lentivirus, respectively.The protein levels of p65 and phosphorylated-p65 were detected by western blot assay and quantitative analysis in A549 cells (A, B, E, F) and NCI-1975 cells (C, D, G, H).About 1 × 10 7 cells infected with the corresponding lentivirus, were implanted subcutaneously into nude mice.The protein levels of p65 and phosphorylated-p65 were detected by western blot assay and quantitative analysis in each xenograft group (I, J). * P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9 .
Figure 9. MiR-1976 targeting NCAPH blocks the activation of NF-kB.The A549 cells and NCI-H1975 cells were infected with the corresponding lentivirus, respectively.The levels of p65 in cytoplasm and nucleus were detected by Laser confocal microscope (A, B).DAPI is used to stain the nucleus.The scale is 50 nm.

Figure 10 .
Figure 10.A diagram shows that MiR-1976 suppresses LUAD via NCAPH/ NF-κB pathway.MiR-1976, which can bind to the NCAPH-3 'UTR terminal, is reduced in LUAD cells, resulting in increased NCAPH mRNA and protein expression in lung cancer.NCAPH overexpression stimulates p65 phosphorylation, which activates the NF-pathway and promotes the development of malignant features in LUAD cells (proliferation, migration, and anti-apoptosis).

Table 1 .
Association between the expression levels of miR-1976 and the clinicopathological features of patients with lung adenocarcinoma.Bidirectional disordered data were tested by pearson Chi-square test.Unidirectional ordered data (T, N, TNM, G) were tested by the rank-sum test of the sequential list (Mann-Whitney U test).TNM tumor node metastasis.